LIGHT pathway in T-cell mediated autoimmunity and infectious disease

Clinical and Applied Immunology Reviews 4 (2004) 367–393

A role for the lymphotoxin/LIGHT Pathway in T-cell mediated autoimmunity and infectious disease Jennifer L. Gommerman* Department of Immunology, University of Toronto, 1 King’s College Circle, Toronto, Ontario, Canada M5S 1A8 Received 15 June 2004; received in revised form 30 July 2004; accepted 30 July 2004.

Abstract It has become increasingly appreciated that signals mediated by the lymphotoxin beta receptor (LTßR), a tumor necrosis factor (TNF) family receptor, mediate various outcomes in both the developing and adult immune systems. In the adult animal, the LTαβ pathway is indispensable for the maintenance of stromal cell networks in the secondary lymphoid tissues. Since these networks are key controlling elements for the positioning of immune cells such as lymphocytes, it was hypothesized that blocking LTßR signaling may be a novel approach for inhibiting pathogenic autoimmune responses. To this end, multiple autoimmune models have been tested in mice that are genetically deficient in LTαβ/LTßR molecules or tested in mice that are treated with LTαβ/LIGHT pathway blocking agents. These studies have revealed an impressive array of autoimmune diseases that are sensitive to LTαβ/ LIGHT pathway inhibition, signifying that blockade of LTßR-mediated signals has exciting clinical potential. A common element to these disease models is the recruitment, activation and migration of autoimmune T-lymphocytes. The mechanism of how blocking signals mediated by the LTßR influences autoimmune T-cell responses remains elusive, in particular because LTßR signaling appears to be critical at diverse biological levels. This review will describe the consequences of blocking LTßRmediated signaling on autoimmune disease models, as well as models of infectious disease and will explore how LTßR activation may regulate T-lymphocyte responses. 쑕 2004 Elsevier Inc. All rights reserved. Keywords: Lymphotoxin pathway; LIGHT; lymphoid micro-architecture; autoimmune models; T-cells Abbreviations: CIA, collagen-induced arthritis; DC, dendritic cells; EAE, experimental autoimmune encephalomyelitis; FDC, follicular dendritic cells; GALT, gut-associated lymphoid tissue; GVHD, graft versus host disease; HEV, high endothelial venules; HSV, herpes simplex virus; HVEM, herpes virus entry mediator; LCMV, lymphocytic choriomeningitis virus; LIGHT, lymphotoxin-like inducible protein that completes with glycoprotein D for binding herpes virus entry mediator on T cells; LN, lymph node; LT, lymphotoxin; LTßR, lymphotoxin beta receptor; LTßR-Ig, LTßR immunoglobulin fusion protein; MZ, marginal zone; NOD, non-obese diabetic mouse; PALS, periarteriole lymphoid sheath; PP, Peyer’s patches; TNF, tumor necrosis factor. * Corresponding author. Tel.: ⫹1-416-978-6959; fax: ⫹1-416-978-1938. E-mail address: [email protected] (J.L. Gommerman). 1529-1049/04/$ – see front matter 쑕 2004 Elsevier Inc. All rights reserved. doi: 10.1016/j.cair.2004.07.001

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1. The molecules of the lymphotoxin/LIGHT pathway The Lymphotoxin alpha (LTα) cytokine was characterized at a molecular level at the same time as the discovery of tumor necrosis factor alpha (TNFα), and the molecules are 28% identical at the amino acid level. Since both secreted cytokines were equally able to induce cell death, LTα was originally called TNFβ [1]. For this reason, it was assumed that LTα and TNFα might play redundant biological functions. However, the later discovery of a second related protein, LTβ, changed this perception. When coexpressed with LTβ, LTα forms a heterotrimeric complex that remains membrane-bound through the transmembrane domain of LTβ, and LTβ on its own does not function in the absence of LTα. This LTαβ complex binds to the TNF family receptor LTßR, but not to the TNF receptors TNFRI and TNFRII [2]. Further complicating this picture is the more recent discovery of a new TNF family ligand, LIGHT, that also binds to the LTßR, as well as its own receptor herpes virus entry mediator (HVEM) and a decoy soluble receptor DcR3 (Fig. 1.) [3]. The expression of cell-surface LTαβ is tightly restricted to hematopoietic cells including activated lymphocytes and a subset of resting B-cells, whereas the LTßR is expressed by nonhematopoietic and myeloid lineage cells, as well as dendritic cells (DC) and follicular TNFα TNFα TNFα

TNFRI TNFRII

LTα LTα LTα

LTβ LTβ LTα

LTβR

HVEM

LIGHT LIGHT LIGHT

DCR3

= TNF/LTα pathway

LTβR-Ig = LTαβ pathway = LT/LIGHT pathway

Fig. 1. Receptor and Ligands of the LTαβ, LIGHT and TNF/LTα pathways. Multiple receptor-ligand pairings are not uncommon in the TNF-receptor family. The TNF pathway consists of TNFRI (TNFR55) and TNFRII (TNFR751) binding to secreted and membrane bound TNFα. The discovery that LTα homotrimer also binds TNFRI and TNFRII initially suggested that LTα may play a redundant biological function. However, the discovery of LTβ ligand delineates and LT pathway from the TNF pathway. LTβR-lg will bind to both LTαβ heterotrimer as well as LIGHT homotrimer thus preventing signaling of each LTβR and HVEM as well as binding of LIGHT to DCR3. LTαβ heterotrimer and LIGHT homotrimer are expressed on hematopoietically-derived cells whereas the LTβR is expressed on mesenchymal stromal cell types and on non-lymphoid hematopoietic cells such as DC and macrophages.

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dendritic cells (FDC) [4,5]. This expression pattern suggests that LT ligands on lymphocytes can signal to DC and to specialized stromal cell elements including FDC. LIGHT is expressed by activated T-cells, as well as on immature DC and HVEM is expressed on lymphocytes, thus providing the potential for intercommunication between T-cells, as well for cross-talk between T-cells and DC [6]. Targeted gene deletion of LTα, LTβ and LTßR, but not LIGHT in mice results in several developmental abnormalities, most notably the absence of lymph node (LN) and Peyer’s patches (PP) [7,8], although there is evidence that LIGHT may play a role in mesenteric LN development in the absence of LTβ [9]. Therefore, analyzing the immunological role of these molecules in knock-out mice has been problematic. To circumvent this problem, a decoy protein consisting of the LTßR fused to an IgG1 Fc domain, LTßRIg, has been employed in a variety of settings in order to block both LTαβ, as well as LIGHT binding to the LTßR without the complicating developmental defects observed in the knockouts [10]. Where this is the case, the term LTαβ/LIGHT pathway will be used. The term LTαβ pathway will alternatively be used to refer to biology that is mediated exclusively by LTαβ signaling of LTßR, independent of the contribution of the LIGHT ligand (Fig. 1). 2. Control of lymphoid microenvironments by the lymphotoxin-ab pathway In order to generate an efficient immune response, the immune system is faced with the challenge of coordinating encounters between antigen presenting cells (APC) and rare antigen specific T-cells. Within the lymphoid tissues, a high degree of spatial and temporal organization exists in order to ensure optimal interactions between lymphocytes and various accessory cells including DC and FDC. The spleen contains white pulp nodules filled with lymphocytes and the white pulp is surrounded by the splenic red pulp. The white pulp is further divided into follicles that are rich in B lymphocytes, and the periarteriole lymphoid sheath (PALS) that is populated by T-lymphocytes. Follicles exist as either primary naı¨ve follicles or as secondary follicles that contain germinal centers (GC), an important microenvironment for the maturation of B-cell humoral responses. Within both primary and secondary follicles is a scaffold of stromal cells, some of which are differentiated stromal cell types called FDCs [11]. These FDC contribute to the GC reaction by providing a source of antigen to B-cells in the form of immune complexes. B-cells that have undergone affinity maturation interact with antigen-bearing FDC in the light zone of the GC and high affinity clones are consequently selected [12]. In the T-cell rich PALS an intricate network of stromal cells as well as interdigitating DC exist to support immune responses. Surrounding the B-cell follicles is a marginal zone (MZ) that provides an entry site for blood-borne antigen and is a compartment that is rich in APC including DC and macrophages, as well as specialized MZ B-cells [13]. Together, these white pulp compartments provide organization and orchestration to the immune response, optimizing interactions between DC and T-cells and between B-cells and FDC, as well as providing opportunities for T-cells to help B-cells in antigen-specific immune responses. To a large extent, LN microarchitecture parallels splenic organization, although with some notable exceptions, such as the absence of a MZ and the fact that LNs are interconnected by a lymphatic drainage system. Currently there are exciting advances taking place that have improved our understanding of cell and antigen flow through the LN, a concept that is central to our understanding of how immune responses are initiated [14].

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Analysis of LTα, LTβ and LTßR deficient mice yielded the seminal discovery that the LTαβ pathway is critical for the development of LN and PP. The mechanism for how LTαβ pathway controls lymphoid tissue development is at least partially explained by the fact that the LTαβ pathway is a key controlling element that mediates the secretion of chemokines such as CXCL13, CCL19 and CCL21, chemokines that recruit immune cells to the developing lymph node anlage [7,8]. While this review will not focus on LTαβ pathway and lymphoid organogenesis, aspects of this paradigm are recapitulated in the adult animal in order to maintain the organization of secondary lymphoid tissue described above. Specifically, Blymphocytes expressing low levels of LTαβ on their cell surface communicate with stromal cells that express LTßR thus triggering the secretion of chemokines and the up-regulation of adhesion molecules. These chemokines bind to their cognate G-protein coupled receptors, and in the case of B-cells, CXCL13 chemokine will bind to B-cell expressed CXCR5 chemokine receptor. Engagement of CXCR5 in turn leads to further up-regulation of LTαβ on the surface of B-lymphocytes, thus inducing a positive feedback loop (Fig. 2.) [15]. This paradigm will be revisited in the context of the maintenance of lymphoid microenvironments in both secondary lymphoid tissues, as well as tertiary lymphoid tissues, such as the ectopic lymphoid structures observed in sites of inflammation.

FDC or other Stromal cell type

Marginal Zone

Follicular B cell

CXCL13

CXCR5

LTβR

PALS Follicle Central Arteriole

Germinal Center

LTα1β2

CXCR5 activation

LTβR-Ig

Fig. 2. Maintenance of splenic organization and chemokine networks by LTβR signaling. Immune cells are retained in specific compartments in the spleen and other secondary lymphoid tissues by virtue of chemokine networks. T lymphocytes and DC express CCR7 and are drawn to CCL19 and CCL21 chemokines secreted by stromal cells within the PALS. CCL19 and CCL21 signal the CCR7 receptor on lymphocytes and DC leading to their chemotaxis towards the PALS. Conversely, B lymphocytes express CXCR5 and are retained within the B cell follicle by virtue of CXCL13 secretion within this compartment. A feedback loop is illustrated in the inset where stromal cell secreted CXCL13 signals CXCR5 on B cells leading to the up-regulation of LTαβ. As B lymphocytes are drawn to the CXCL13 secreting stromal cells, LTαβ on the B lymphocytes in turn signals LTβR on the resident stromal cells. The constitutive signaling of LTβR is required to maintain these chemokine gradients as well as the differentiation status of FDC in primary and secondary follicles. LTβRlg treatment circumvents this feedback loop leading to splenic disorganization.

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2.1. The germinal center microenvironment The administration of LTßR-Ig to adult wild-type mice allowed visualization of the most dramatic example of this paradigm. Within days following a single dose of LTßR-Ig, markers identifying FDC in the B-cell follicles of mouse spleens were shown to rapidly disappear. It is unclear whether these cells are dedifferentiating into less specialized stromal cell types or are dying in situ, but this observation was particularly stunning given that FDC were thought to be a sessile, terminally differentiated cell type harboring antigen for protracted periods of time [16]. Presumably FDC express the LTßR and require signaling via B-cell expressed LTαβ heterotrimer in order to be maintained in a fully mature, differentiated state (Fig. 2.). FDC are a critical component of the architecture of GC in the B-cell follicle, and not surprisingly, GC are either not formed or formed inefficiently in LTßR-Ig treated mice [16]. The same observation was recapitulated in Cynomolgus macaque primates along with the demonstration that antigen trapping on FDC is also LTßR-Ig sensitive. Furthermore, the presence and function of FDC can be restored within two to three weeks after the level of LTßR-Ig in the serum has dropped below a concentration of 5 µg/ml, illustrating the dynamic nature of FDC [17]. Thus, LTßR signals appear to have similar effects in higher order species and the expectation is that LTßR-Ig treatment will similarly perturb the maintenance of human lymphoid architecture. Although many studies have highlighted the role of FDC in displaying antigen immune complexes for selection of high affinity B-cell clones [12], it is debatable whether this function is absolutely essential for the generation of high affinity class-switched antibody responses to antigens [18,19]. Measurement of antibody affinities in LTβ-deficient immunized mice revealed reduced levels of high affinity antibody when antigen is administered at low doses [20], however, immunization with high doses of antigen yields a relatively normal antibody responses in LTα and LTßR ⫺/⫺ mice [21,22]. In addition to the antigen dose differences, Wang et al. [23] have shown that, in contrast to immunization with soluble antigen in adjuvant, administration of antigen coupled to sheep red blood cells (SRBC) without adjuvant leads to a dramatic reduction in high affinity antibodies in LTα ⫺/⫺ mice. Therefore, under some conditions, the quality of antibody responses in LTαβ pathway deficient animals is compromised, presumably due to the absence of FDCmediated selection in the GC. 2.2. Splenic organization in LTßR-Ig treated mice Other aspects of splenic organization such as the proper segregation of T-cell and B-cell areas and the organization of the MZ compartment are also sensitive to LTßR-Ig treatment [24,25]. In the spleens of LTαβ and LTβR-deficient and LTßR-Ig treated mice, there is a decrease in CCL19, CCL21 and CXCL13 chemokines in the spleen [26]. Triggering of CXCR5 on B-cells by CXCL13, which is secreted by FDC induces B-cell homing to the follicles [15]. CCL19 and CCL21 are produced constitutively by stromal cells within the splenic PALS and T-cell areas of LNs, and CCL21 can also be expressed by HEV and lymphatic endothelium [27,28]. These two ligands bind to the CCR7 chemokine receptor expressed on T-cells and DC and mediates migration of these cells to the T-cell rich regions of lymphoid tissues [29]. Therefore, these constitutively expressed chemokines control immune cell positioning and migration, and the reduction of these chemokines in LTßR-Ig treated mice

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likely accounts for the poor segregation of T-cells and B-cells in these animals. In addition, it is has been shown that the number of splenic DC in LT-deficient mice is greatly diminished and accompanied by disorganized positioning of remaining DC within the spleen. Since CCL19 and CCL21 chemokines bind to CCR7 expressed on DC, down-regulation of these chemokines by LTßR-Ig treatment represents a likely mechanism for the reduced DC numbers in treated mice [30]. With respect to the MZ compartment, expression of MAdCAM-1 on the MZ sinus is exquisitely LT-sensitive. Furthermore, a significant reduction of metallophilic macrophages, MZ macrophages and MZ B-cells was observed in LTßR-Ig treated mice [25,31,32]. The mechanism for why macrophages in the MZ are affected by LTαβ pathway is unclear, however LTαβ pathway may control the retention of MZ B-cells in the MZ via the up-regulation of integrins on an unidentified MZ-resident stromal cell [33]. Therefore, many aspects of splenic organization depend on constitutive signaling of the LTßR. It should be noted, however, that some aspects of the splenic microenvironment such as the expression of chemokines by follicular stromal cells and MAdCAM-1 expression on the MZ sinus are also TNF-dependent [26,34,35]. 2.3. Thymic organization The interaction between thymic epithelial cells and thymocytes is important for generating a mature T-cell repertoire. Boehm et al. [36] have found that LTßR signaling on thymic medullary epithelial cells (MEC) is required for their normal differentiation, and LTßR ⫺/⫺ mice, as well as LTßR-Ig treated mice have reduced numbers of thymic MECs. In the case of LTßR ⫺/⫺ mice, this phenotype was associated with autoantibody production to antigens present in salivary gland, pancreas and stomach. In addition, inflammatory infiltrates have been observed in the lungs of LT-deficient mice [37]. An autoimmune regulator (AIRE) has been shown to regulate the promiscuous gene expression of various tissue-specific antigens, and expression of both AIRE and tissue-specific genes in MECs is important for thymic selection of developing T-lymphocytes. LTßR ⫺/⫺ mice were found to have normal expression of AIRE, as well as tissue specific antigens such as salivary protein 1 in thymic MECs. Therefore, presumably loss of tolerance in LTßR ⫺/⫺ mice is independent of AIRE expression and is due to other developmental factors. In agreement, Kajiura et al. [38] show that AIRE expression in LTα ⫺/⫺ mice is normal, but curiously the development of immunoregulatory CD4⫹CD25⫹ T-cells is significantly impaired, thus providing a possible explanation for the autoimmune phenomenon that is associated with LT-deficient mice. 2.4. Mucosal lymphoid microenvironments PP and mesenteric LN are examples of organized lymphoid compartments of the gut. In addition to the maintenance of specialized stromal/reticular cell types in the spleen, the LTαβ pathway regulates some aspects of cellular organization within the mucosal system. Specifically, the M-cell content of PP and the cellularity of PP and colonic patches are significantly diminished with LTßR-Ig treatment [39,40]. In addition, isolated lymphoid follicles that develop postgestationally and are dependent on the presence of luminal microbederived stimuli require LTßR-signaling [41]. Thus, although PP and LN are clearly dependent

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on LTßR signals for their proper development during embryogenesis, isolated lymphoid follicles are an example of an organized lymphoid structure that is dependent on LTßR signals after gestation. The counter example to the isolated lymphoid follicle observation is the nasal-associated lymphoid tissue (NALT), which also develops after birth but is not dependent on LTßR-mediated signals as NALT is present, albeit in reduced size, in each LTα ⫺/⫺, LTβ ⫺/⫺ and LTßR ⫺/⫺ mice [7]. Finally, there is a clear role for LTαβ pathway in regulating the lamina propria microenvironment that is important for IgA production, as signaling of LTßR on lamina propria stromal cells mediates IgA production in the gut [42,43]. Overall, however, the mechanisms for the role for LTαβ pathway in the context of the gut and the chemokines involved are not as well delineated as for the spleen. Taken together, the administration of LTßR-Ig blocking agent to adult mice has led to the discovery that several reticular networks in the lymphoid organs are under LT control and that elimination of these networks results in dramatic dysregulation of chemokine gradients that are required for the proper positioning of immune cells. This paradigm has led to the hypothesis that LTßR-Ig treatment and consequently dysregulation of the lymphoid microarchitecture is a novel approach for treating autoimmune disease, since it is in the lymphoid organs that naı¨ve lymphocytes encounter antigen [44]. 3. The lymphotoxin-ab pathway and ectopic lymphoid structures Ectopic lymphoid structures or tertiary lymphoid tissues have been observed in settings of chronic inflammation and the emergence of these tertiary lymphoid aggregates has been correlated with human disease. Specifically, these structures have been noted in the gut in Crohn’s patients, in the synovium of rheumatoid arthritis patients, in the salivary glands of Sjo¨gren’s syndrome patients, in the thyroid in autoimmune thyroiditis patients and other autoimmune conditions such as myasthenia gravis [45]. In many of these human clinical examples, FDC are present and help establish ectopic GC reactions that likely contribute to the production of autoantibodies [46]. A convenient way of analyzing which molecules are involved in induction of ectopic lymphoid structures is to aberrantly express candidate proteins in peripheral tissues. To this end, transgenic mice have been generated that ectopically express candidate genes under the control of the rat insulin promoter (RIP) so that the development of ectopic lymphoid structures can be visualized within the pancreas. Accordingly, given their roles in LN development during embryogenesis, LTα and LTβ, as well as each of the chemokines CXCL13, CCL19, CCL21 and CXCL12 have been analyzed for their role in inducing ectopic lymphoid structures. The first of these studies was executed by Kratz et al. [47] who generated RIP-LTα mice. These mice develop organized and segregated B-cell and T-cell regions, primary and secondary follicles and evidence of high endothelial venule (HEV) structures within the pancreas. A subsequent elegant study by Ruddle and colleagues showed that transgenic coexpression of both LTα and LTβ under the control of RIP induced significantly larger infiltrates than LTα alone, and this correlated with the presence of peripheral node addressin (PNAd) on the abluminal aspect of the HEV. This presumably permits enhanced lymphocyte trafficking to the ectopic lymphoid aggregate by binding to L-selectin on naı¨ve lymphocytes. An HEV-restricted sulfotransferase that is known to regulate the

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luminal expression of PNAd was found to be regulated by LTαβ expression, thus providing an important cog in the wheel of ectopic lymphoid structure development [48]. In this same study, an increase in the chemokines CCL21, CCL19 and CXCL13 was observed. RIP-driven transgenic expression of each of these chemokines also induces organized lymphoid aggregates with differing degrees. RIP-CXCL13 mice develop ectopic lymph node-like structures containing B-cell areas, T-cell areas, HEV and stromal cells, but not GC. Importantly, these structures could be dissolved by LTßR-Ig treatment [49]. Ectopic expression of CCL21 in the pancreatic islets also induced the infiltration of T-cells, B-cells and DCs as well as induction of HEVs [50,51]. To compare the activities of CCL21, CCL19 and CXCL12, Luther and colleagues [52]directly compared RIP-transgenic mice specific for each chemokine. They found that pancreatic CCL21 expression induced larger and more organized aggregates than CCL19 ectopic expression whereas CXCL12 expression in the pancreas resulted in qualitatively different aggregates that were dominated by plasma cells, B-cells and DC. Interestingly, LTßR-Ig treatment did not affect the cellularity or the composition of infiltrates in CCL21-RIP mice, but the expression of PNAd and MAdCAM on HEVs within the infiltrated islets was strongly reduced. Since LTßR-Ig treatment failed to reduce the existing size of pancreatic ectopic lymphoid structures, it was hypothesized that induction of LTαβ pathway may lie downstream of aberrant CCL21 expression, and that LTαβ pathway may be important for recruitment of naı¨ve lymphocytes by regulating the expression of addressins on HEV. In agreement with this hypothesis, CCL21 chemokine and to a lesser extent, CCL19 can induce the expression of LTαβ on naı¨ve lymphocytes in vitro. In the human disease scenario, expression of LTβ, CXCL13 and CCL21 have all been noted in sites of chronic inflammation [45], however the sequence of which cytokine/chemokine comes first in the disease pathogenesis process in humans is not clear. It has not been directly proven that the establishment of ectopic lymphoid structures is a key step toward the loss of tolerance to autoantigen and the development of human autoimmune disease. However, since a potent immune response thrives on optimal organization of lymphocytes, adequate antigen presentation and the presence of antigen-reactive lymphocytes [44], it makes sense that an ectopic lymphoid aggregate would perpetuate the autoimmune process. Thus, the conversion of lymphocyte aggregates into bonafied ectopic lymphoid structures may be a critical step toward full-blown autoimmunity in humans [45]. This is one reason why LTαβ/LIGHT pathway inhibitors have been logical candidates for clinical treatment of autoimmunity. However, the effects of LTßR-Ig are not limited to the dissolution/modulation of tertiary lymphoid tissue since LTαβ/LIGHT pathway likely acts at multiple checkpoints in the autoimmune cascade (Fig. 3). 4. The lymphotoxin-ab pathway and rodent models of autoimmunity Since LTαβ pathway clearly plays an important role in regulating lymphoid microenvironments, several groups have investigated its role in initiating or propagating autoimmune responses. An impressive array of rodent autoimmune disease models has been tested either in the context of LT-deficient mice or by treatment with LTßR-Ig, and here we will focus exclusively on cases where LTαβ and/or LIGHT pathways are inhibited in adult rodents where LTαβ/LIGHT related developmental abnormalities are not an issue (Table 1).

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4.1. Inflammatory bowel disease (IBD) LTαβ/LIGHT pathway plays a pivotal role in gut immunology for several reasons [53]. First, LTßR signaling is critical for the development of PP (reviewed in [7]). Moreover, the cellularity of PP and colonic patches, in particular the accumulation of B-cells within these structures, is controlled by LTαβ pathway in a postgestational manner [40]. Second, isolated lymphoid follicles are a type of tertiary lymphoid structure that forms in the gut and their formation requires LT signaling [41]. Third, transgenic overexpression of LIGHT induces an IBD-like condition in mice [54,55]. Finally, colonic samples from patients with ulcerative colitis (UC) and Crohn’s disease (CD) show evidence of infiltrating plasma cells and CD4⫹ T-cells that express very high levels of LTβ [56]. Therefore, given the important role of LTαβ/LIGHT pathway in gut microenvironments and IgA production and the correlation of LT expression in diseased gut tissue, several groups have assessed the therapeutic efficacy of LTßR-Ig in rodent models of IBD. Administration of LTßR-Ig was found to profoundly reduce colitis in two models of IBD that require T-cell transfer. In the first model, gut inflammation is triggered by the transfer of CD45RBhi T-cells into immunodeficient SCID mouse recipients, inducing an uncontrolled expansion and activation of CD45RBhi cells that in turn triggers inflammation. LTßR-Ig treatment significantly reduced inflammation and prevented wasting disease in this model. In a second transfer model T-cell/natural killer cell deficient tgε26 mice are transplanted with normal bone marrow, and transplanted CD4⫹ T-cells expand and induce gut inflammation. This model was also inhibited by LTßR-Ig treatment, as well as anti-TNFα treatment [57]. Thus, LTαβ/LIGHT pathway inhibition was successful in treating two models of IBD that depend on the transfer of T-lymphocytes. Other models of IBD have also been assessed. Oral administration of dextran sulfate sodium induces chronic colitis in mice and strong LTβ expression is found in the colons of these treated mice. This model of chronic colitis was found to be inhibited by LTßR-Ig treatment and the therapeutic efficacy was accompanied by decreased levels of inflammatory cytokines TNFα, IL-1 and IL-6, as well as decreased expression of the MAdCAM-1 addressin in the gut [56]. Hapten-induced murine colitis is initiated by treatment with trinitrobenzene sulfonic acid in mice. To evaluate a Th2 dominant form of this colitis, trinitrobenzene sulfonic acid was administered to IFN-γ deficient mice and these were then treated with LTßR-Ig. LTßRIg treatment resulted in a reduction in PP and colonic patch size with an accompanying decrease in DC in these organs. Decreased disease was observed with LTßR-Ig treatment only in IFN-γ deficient mice, thus suggesting that LTßR-Ig treatment is efficacious in a Th2dominant form of colitis [40]. Therefore, in four different models of IBD, LTßR-Ig has shown to inhibit disease progression implying that LTαβ/LIGHT pathway is involved in the progression of IBD. Since LTαβ/LIGHT pathway is relevant to many aspects of gut immunology [53] it will be of interest to dissect the mechanism of action of this therapeutic efficacy. 4.2. Diabetes T-cells are important in the pathogenesis of diabetes. Two studies evaluating the effect of LTαβ/LIGHT pathway inhibition on insulin dependent diabetes mellitus (IDDM) were performed using the non-obese diabetic mouse strain (NOD). In the first, mice expressing a

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Table 1 Effect of LT αβ/LIGHT pathway inhibition on rodent models of human disease Animal Model

Comments

Experimentally induced Monophasic EAE in Lewis Rat [74] Relapsing-Remitting SJL EAE [74]

Only prophylactic treatment was successful Inhibition with anti-LTβ equally effective as LTβR-Ig Acute phase was unchanged, relapses were inhibited

Uveitis

Experimentally induced Uveitis in Lewis Rat [76]

Could not inhibit disease induced by adoptive transfer of pre-primed autoimmune T cells

Transplantation

Intestinal allograft [64] Acute GVHD [61] Acute GVHD [62] Minor mismatch skin GVHD [63] Cardiac allograft [65]

Inhibition with anti-LTβ-equally effective as LTβR-Ig Inhibition with both LTβR-Ig and anti-LIGHT antibody Complete inhibition of GVHD with LTβR-Ig plus anti-CD40L Reduced skin lesions and decreased ICAM-1 expression with LTβR-Ig Prolonged allograft survival with DcR3-lg (LIGHT inhibition)

Insulin-dependent diabetes

NOD NOD NOD NOD

LTβR-Ig treatment prevented and reversed insulitis LTβR-Ig transgene expression prevented diabetes but not insulitis HVEM-Ig treatment reverses diabetes DcR3-Ig transgene expression prevents diabetes

Inflammatory Bowel Disease

CD45RBhi colitis [57] CD3ε-transgenic colitis [57] TNBS-induced colitis [40] DSS-mediated chronic colitis [56]

Suppression of inflammation and wasting disease Efficacy of LTβR-Ig was equivalent to anti-TNF Efficacy with LTβR-Ig only observed in a Th2-setting LTbR-Ig treatment reduced inflammatory cytokines and MAdCAM-1 expression in the gut

Rheumatoid arthritis

Collagen-induced arthritis [77]

Reduced disease both prophylactically and therapeutically

mouse mouse mouse mouse

[58] [32] [55] [59]

Note: Listed here are cases where LTαβ/LIGHT pathway inhibition prevented or ameliorated disease in rodent models of autoimmunity. Disease models that were performed in LIGHT, LTα, LTβ or LTβR knock-outs have been excluded, and only cases where LTαβ/LIGHT pathway inhibition has been performed in adult animals are listed. In some cases, reagents that inhibit only the LTαβ pathway (grey boxes) or conversely, only the LIGHT pathway (empty boxes) , have also been tested. LTαβ/LIGHT pathway inhibition did not have an effect in MOG ⫹ PT× EAE [74], in Th1-mediated colitis [40], and in antibody/LPS induced acute arthritis [77].

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Human Disease Multiple sclerosis

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neutralizing LTßR-Fc chimeric transgene were back-crossed onto the NOD mouse background [32]. Examination of the pancreatic islets revealed that expression of the fusion protein had no effect on insulitis although alterations in splenic architecture were readily apparent, indicating effective LTαβ/LIGHT pathway inhibition. However, in spite of observed insultitis, diabetes development was prevented. Moreover, in vitro T-cell proliferation and secretion of IFN-γ in response to the pancreatic autoantigen, glutamic acid decarboxylase (GAD), was attenuated in fusion protein expressing mice. A second study has also shown a therapeutic benefit of LTßR-Ig pharmacological treatment in preventing disease development in NOD mice, although in this study insulitis was completely prevented. Indeed, these authors showed that moderate to severe insulitis could be reversed at 12 weeks of disease [58]. It is unclear why these studies show differences in the development of insulitis and the differences could reflect the level of LTßR-Ig fusion protein in the circulation. In the case of the transgenic expression of LTßR-Ig, when concentrations of fusion protein dropped below 2 µg/ml in the serum, diabetes resumed and other microarchitectural elements in the lymphoid organs were restored. Nonetheless, both studies support the hypothesis that LTαβ/LIGHT pathway might be involved in autoimmune T-cell responses. However, at what point T-cells were sensitive to LTßR-Ig treatment, and whether inhibition of disease was due to exclusive LTαβ signaling and/or LIGHT signaling through LTßR was not resolved. A subsequent study by Wang et al. [55], however, demonstrated that administration of HVEM-Ig to prediabetic NOD females could prevent the development of IDDM, suggesting some activity of LIGHT in the development of diabetes independent of LTαβ signaling of the LTßR. A more recent study lends support to the hypothesis that LIGHT is important in diabetes pathogenesis. Sung et al. [59] expressed the decoy receptor 3 (DcR3), a soluble TNF-family receptor under the control of the human insulin promoter in NOD mice so that DcR3 would be expressed specifically in pancreatic β cells. DcR3 binds to LIGHT, Fas-ligand and TNFlike molecule 1A, therefore local expression of DcR3 in the pancreas will inhibit these pathways. Expression of the DcR3 transgene in NOD mice significantly protected these mice from both spontaneous and cyclophosphamide-induced diabetes. These experiments do not discriminate whether LIGHT and/or Fas pathway are important in diabetes pathogenesis, but the combination of this experiment with the previous LTßR-Ig experiments is suggestive of a role for LIGHT in the diabetes disease process. Importantly, the study by Sung et al. [59] demonstrated that T-cells from DcR3 transgenic NOD mice do not exhibit any intrinsic defects, therefore if the effect on diabetes in these mice is due to LIGHT, this effect is restricted to the effector phase of the disease. 4.3. Transplantation Given that both LIGHT and LTαβ are expressed on activated T-cells [4], LIGHT and HVEM are expressed on immature DC [3] and LTßR is expressed on DC [5], there is the potential for communication between activated T-cells and antigen presenting DC in the initiation of immune responses. Graft versus host disease (GVHD) is an acute example of T-cell recognition of alloantigens, and GVHD remains a significant clinical complication in organ and bone marrow transplantation. Since LIGHT was first described as a costimulatory molecule, [60] the effect of LIGHT blockade in an acute GVHD response was tested. In this study,

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Tamada et al. [61] were able to diminish GVHD by administration of LTßR-Ig, as well as anti-LIGHT antibodies. This was accompanied by reduced CD8⫹-mediated cytolytic T-cell responses to host allogeneic antigen. This finding indicated that LIGHT was involved in GVHD, and subsequent studies of the LIGHT ⫺/⫺ mice have confirmed this hypothesis [9]. However, the study by Tamada et al. [61] did not rule out the involvement of LTαβ binding to LTßR-Ig in GVHD since LTßR-Ig was also efficacious. In a subsequent study, concurrent administration of LTßR-Ig and anti-CD40L completely abrogated GVHD, thus the combination of these two inhibitors could yield a potent therapy for this clinical problem [62]. LTßR-Ig treatment was also tested in a different model of GVHD whereby bone marrow carrying a minor histocompatibility mismatch is transplanted to recipient mice. Transplanted recipient mice develop a psoriasis-like skin condition in response to the mismatched graft. Mice treated with LTßR-Ig did not exhibit skin lesions and T-cell trafficking to the skin was prevented. In addition, the expression of the ICAM-1 adhesion molecule in the skin was significantly decreased with treatment, suggesting a possible mechanism for the inhibition of this form of GVHD with LTßR-Ig treatment [63]. In addition to GVHD, other transplantation settings have been explored. In particular, intestinal allograft survival was assessed by Guo et al. [64]. These authors found that LTßR-Ig administration prevents rejection of allografts in a setting where the rejection was entirely mediated by CD8⫹ T-cells. Importantly, this study showed that an anti-LTβ monoclonal was also an effective inhibitor of intestinal allograft rejection, thus implicating LTαβ pathway independent of LIGHT. The increased allograft acceptance was accompanied by decreased levels of monokine-induced IFN-γ (MIG) and CCL21 chemokine gene expression within the allografts [64]. Thus, inhibition of LTαβ pathway may result in the decrease of IFN-γsensitive chemokine levels and consequently prevent migration of effector cells to inflamed tissue. A separate study was able to identify a potential role for LIGHT independent of LTαβ in mediating cardiac graft survival by treating mice with DcR3-Ig, which will block LIGHT-mediated signaling. However, a potential caveat, as mentioned in the aforementioned diabetes study, is that this fusion protein will block both LIGHT and Fas-mediated events [65]. Nonetheless, survival of cardiac allografts is enhanced in LIGHT knock-out mice, suggesting that the actions of DcR3-Ig may indeed be through the LIGHT pathway [66]. Taken together, these studies have revealed independent roles for both the LTαβ and the LIGHT pathways in regulating allograft rejection and point to a central role for these molecules in affecting T-cell responses. 4.4. Multiple sclerosis Experimental autoimmune encephalomyelitis (EAE) is a rodent model of multiple sclerosis (MS) that can be executed on genetically susceptible backgrounds. EAE has been induced in LTα ⫺/⫺, LTβ ⫺/⫺, TNFα ⫺/⫺ and TNFα/ LTα ⫺/⫺ mice to dissect the relative contributions of LTαβ and TNF pathways to MS [67–70]. The study of EAE in these mice has been complicated by the fact that the LTαβ pathway-deficient mice do not have LN and also that both LTα and LTβ genes are located within the mouse major histocompatibility complex (MHC) [71]. Since EAE models are often performed on susceptible strains, back-crossing from the 129 strain becomes problematic [72]. LN-competent bone marrow chimeric mice

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using bone marrow from C57BL/6 LTα ⫺/⫺ mice were generated in order to circumvent these problems, and this rigorous approach revealed that LTα did not play a role in EAE [69]. However, in this context, an EAE model requiring the administration of pertussis toxin (PTx) was executed. PTx is used to induce blood-brain barrier permeability in C57BL/6 mice, however it has the additional complication of inhibiting G-protein coupled chemokine receptor signaling [73]. As described earlier, levels of CXCL13, CCL19 and CCL21 chemokines in the secondary lymphoid tissues are under LT control [26]. Therefore, PTx-dependent EAE models need to be compared with other models that do not require this agent. The role of LTßR signaling in EAE was revisited using LTßR-Ig to inhibit the LTαβ/LIGHT pathway in the adult animal and by comparing EAE models that do and do not require PTx administration [74]. LTαβ/LIGHT inhibition resulted in prevention of disease in an acute rat model, and prevention of relapses in a relapsing-remitting mouse model. In parallel with these results, in vitro T-cell responses to encephalogenic peptides were profoundly depressed if T-cells were derived from LTßR-Ig treated animals. It is notable that direct depletion of activated T-cells by LTßR-Ig does not contribute to the mode-of-action, since an aglycosylated form of LTßR-Ig that cannot bind to Fc receptors was similarly effective. However, LTßRIg treatment had no effect on myelin oligodendrocyte glycoprotein (MOG)-induced EAE, which, unlike the other two models, requires the administration of PTx. Therefore, by testing a variety of models, a role for LTαβ/LIGHT pathway in EAE was observed. Interestingly, the same anti-LTβ monoclonal antibody that was effective at preventing intestinal graft rejection was similarly effective in preventing rat EAE, thus providing a second example of the involvement of LTαβ pathway in autoimmune disease independent of LIGHT. It is unclear why the acute rat EAE model was completely inhibited by LTßR-Ig treatment whereas the acute phase of the relapsing-remitting EAE model was unaffected. Perhaps the effector mechanisms responsible for monophasic EAE compared to the acute phase of relapsing EAE differ, and hence are modulated differently by LTßR-Ig. In addition, rat EAE could not be prevented if LTßR-Ig was administered after disease onset. It is assumed that in the context of the Lewis rat, once T-cells are activated and polarized to a Th1 phenotype, the proinflammatory effector response is beyond recall. Therefore, LTαβ/LIGHT pathway inhibition would need to occur during the priming of T-cells in order to have an impact on their effector function later on. 4.5. Other autoimmune models Additional evidence for the therapeutic efficacy of LTßR-Ig is still emerging. A recent study demonstrated that LTα deficient mice do not develop experimentally induced myasthenia gravis [75]. In addition, Shao et al. [76] have shown that LTßR-Ig treatment prevents the induction of uveitis in Lewis rats by immunization with a uveitogenic peptide, R16. Similar to other studies mentioned here, the T-cell responses to R16 were strongly suppressed in LTßR-Ig treated mice. Interestingly, LTßR-Ig treatment could not prevent uveitis mediated by adoptive transfer of R16-reactive T-cells suggesting that there are key events that are LTαβ/ LIGHT-dependent at the initiation of disease, at least in the Lewis Rat. Models for rheumatoid arthritis have also been tested in the context of LTαβ/LIGHTinhibition, and collagen-induced arthritis (CIA) is one model that has been extensively

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explored. In this model, mice are immunized with type II collagen and develop an autoreactive antibody response specific for mouse collagen that induces inflammation in the joints that is complement-mediated. LTßR-Ig prophylactic administration prevented the induction of CIA and therapeutic treatment was effective at arresting preestablished disease. Dissecting how LTßR-Ig prevents or arrests CIA has been problematic due to the multiple disease mechanisms involved in this model. The study by Fava et al. [77] showed that LTßR-Ig treatment resulted in a reduction of collagen-specific antibody concomitant with defective germinal center development in the draining LN. Although T-cell responses to type II collagen in the draining LN appeared normal, overall levels of IFN-γ in the serum of treated mice were reduced. Therefore, in the context of CIA, it is less clear what effect LTßR-Ig treatment has on T-cell responses. Collectively, these studies strongly suggest that LTαβ/LIGHT pathway plays a critical role in the induction of autoimmune responses, and in many of these models there is a clear role for LTαβ/LIGHT pathway in controlling the autoreactive T-cell response. In a few cases, the activities of the LTαβ pathway have been distinguished from the contribution of LIGHT, such as in the intestinal allograft and the acute EAE autoimmune models. The multiple biological roles of LTαβ and LTαβ/LIGHT pathway are illustrated in Fig. 3. The complexity of this picture has hampered efforts to understand why the LTßR-Ig reagent is effective in an impressive array of autoimmune disease models. Clues to how this pathway regulates the autoimmune response can be derived from analyzing responses to well characterized antigens. These will be reviewed in the next sections in order to arrive at a potential mechanism for the therapeutic efficacy of LTßR-Ig. 5. The lymphotoxin-ab pathway and infection Analysis of the influence of LTαβ/LIGHT pathway on immune responses to pathogens is important in order to determine whether LTßR-Ig treatment will result in immunosuppression and susceptibility to infection. In addition, these analyses provide further information on how LTαβ/LIGHT pathway influences lymphocyte responses. Here, only infectious agents relevant to T-lymphocyte responses will be discussed, therefore cases of susceptibility to infections due to the developmental effects of the absence of LTαβ pathway molecules will be excluded. Moreover, only infectious diseases affected by LTαβ/LIGHT but not TNF/LTα pathway will be discussed and displayed in Table 2. 5.1. LCMV Lymphocytic choriomeningitis virus (LCMV) infection of mice is a model that is used to analyze immune responses to acute infection. Two different strains have been examined in the context of the LTαβ pathway. A strong anti-viral cytotoxic T-lymphocyte (CTL) response is elicited in mice infected with LCMV Armstrong stain (LCMV-ARM), and this CTL responses effectively clears the virus at 14 days postinfection. LTβ ⫺/⫺ mice that were infected with LCMV-ARM exhibited poor CTL activity at all inoculate doses tested. At 60days postinfection, there was a 140-fold reduction in LCMV-ARM reactive T-cells, indicating that the memory response to LCMV-ARM was similarly affected. Accordingly, clearance of LCMV-ARM from the serum of LTβ ⫺/⫺ mice was strongly impaired. In the case of

1. De-differentiation of specialized stromal elements and disruption of constitutive chemokine networks

LTβR-Ig 7. Reduced cellularity of PP and ablation of isolated lymphoid follicles in the gut

3. Inhibition of LIGHT-mediated co-stimulation of T cell responses

4. Impaired DC migration to secondary 6. Decreased secretion of inducible lymphoid tissues and DC positioning chemokines associated with inflammation (i.e., MIG) 5. Impaired T cell responses to virus and auto-antigens including reduced IFN-γ secretion

Fig. 3. Biological effects of inhibiting the LTαβ/LIGHT pathway. Administration of LTβR-lg prevents the binding of both LIGHT and LTαβ to their receptors. This agent has been implicated in a number of biological outcomes in vivo and these effects are represented here schematically. The gery region denotes activities that are relevant to the initiation of the immune response whereas inhibition of the activities in the white section affect the inflammatory/effector phase of the autoimmune cascade, although in reality these two phases may not be readily segregated. Inhibition of outcomes #1 and #5 may effect both the initiation of the immune response as well as the inflammatory phase.

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2. Prevention of GC formation and ablation of existing GC. Effects on affinity maturation of antibody responses are variable.

8. Dissolution of ectopic lymphoid structures

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the highest inoculation dose, clearance of LCMV in the brain and kidneys of LTβ ⫺/⫺ was also impaired, but this was not the case at lower inoculation doses [78]. To rule out the absence of LN as the reason for impaired LCMV responses, this study went on to show that transplantation of LTβ ⫺/⫺ bone marrow into LTβ ⫹/⫹ recipients (LTβ ⫺/⫺→ LTβ ⫹/⫹) was accompanied by poor LCMV-ARM clearance. In these bone marrow chimeras, LN are present, but the splenic MZ is disorganized. Since the splenic MZ is rich in MZ macrophages, metallophilic macrophages (MM) and DC, all of which produce IFN-α/β in response to virus infection, it is quite possible that the disrupted splenic microenvironment in LTβ ⫺/⫺ and LTβ ⫺/⫺→LTβ ⫹/⫹ chimeras is responsible for impaired CTL responses to LCMV-ARM. However, the study does not rule out a direct role for LT expression on activated lymphocytes independent of the microenvironment. A second study revisited the role of LTßR signaling in LCMV-ARM responses, in this case using LTα ⫺/⫺ mice. Similar to the LTβ ⫺/⫺ mice, LTα ⫺/⫺ mice exhibited profound defects in LCMV-ARM responses. This was demonstrated by a reduced number of LCMVARM specific CD8⫹ T-cells, defective CTL activity and IFN-γ secretion and impaired viral clearance. Interestingly, the defect in IFN-γ secretion was more profound than the actual decrease in LCMV-ARM specific CD8⫹ T-cells, suggesting that there are LCMV-ARM specific CD8⫹ T-cells in LTα ⫺/⫺ mice that are functionally unresponsive. This study aimed to dissect the importance of LTα expression on T-cells for the LCMV-ARM response. Adoptive transfers of LTα ⫺/⫺ splenocytes into LN-competent LTα ⫹/⫹ mice (LTα ⫺/⫺ → LTα ⫹/⫹) were performed and the recipients were infected with LCMV-ARM. In contrast to the bone marrow chimera results of Berger et al. [78], this study showed that recipients of LTα ⫺/⫺ splenocytes had normal LCMV responses. The authors went on to demonstrate that the T-cell responses were not host derived, but rather originated from the adoptively transferred LTα ⫺/⫺ lymphocytes. The same group went on to analyze a lethal LCMV model (LCMV virus clone 13) where infected mice succumb to pulmonary distress by day five postinfection and death at day seven that is paradoxically mediated by the LCMV-specific CD8⫹ T-cells. Interestingly, treatment with LTßR-Ig prevented pulmonary distress and significantly improved survival of LCMV13 infected mice whereas TNF blockade had no effect. This could be accomplished by a single-dose treatment after establishment of viral infection on day three. The number of LCMV-13 specific CD8⫹ T-cells was significantly reduced in LTßR-Ig treated mice [80]. In this case, the relative contribution of LT expression by T-lymphocytes versus LT effects on the microenvironment was not discerned. A single injection of LTßR-Ig results in elimination of FDC networks and MAdCAM-1 expression on the MZ sinus of the spleen within one week of treatment. However, three weeks of treatment are needed for disruption of splenic T/B lymphocyte organization, three to four weeks are required for total eradication of MZM and MM [25], and 10–15 days of treatment resulted in splenic DC reduction [30]. Therefore, since four days of LTßR-Ig treatment successfully prevented pulmonary distress induced by LCMV-13 infection, contributions of LT/LIGHT independent of these microenvironmental compartments may also be relevant for LCMV-13 clearance. 5.2. Other viruses Other viruses have been tested in LTα ⫺/⫺ mice with differing results. Kumuraguru et al. [81] showed that intradermal or intramuscular injection of differing doses of HSV-1

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Table 2 Effect of LTαβ/LIGHT pathway inhibition on models of infectious disease Infectious agent and hostgenotype

Route of administration

LCMV-ARM → LTβ⫺/⫺ mice [78]

Intraperitoneal

LCMV-ARM → LTβ⫺/⫺ chimeras [78]

Intraperitoneal

LCMV-ARM → LTα⫺/⫺ mice [79]

Intraperitoneal

LCMV-ARM → Recipients of adoptive transfer of LTα⫺/⫺ splenocytes[79] LCMV-Cl. 13 → LTβR-Ig treated mice [80] HSV-1 → LTα⫺/⫺ mice [81]

Intraperitoneal

Intravenous

Influenza type A → LTα⫺/⫺ mice [82]

Intranasal high and low dose

M. tuberculosis → LTβ⫺/⫺ chimeras [84] M. tuberculosis → LTα⫺/⫺ chimeras [84] M. tuberculosis → LTβR chimeras [86] M. bovis → LTβR-Ig treated mice [85] L. monocytogenes → LTβR⫺/⫺ mice[81] T. gondii → LTα⫺/⫺ chimeras [87]

Aerosol

T. brucei → LTα⫺/⫺ mice [81]

Intradermal or Intramuscular

Aerosol Intranasal Intraperitoneal Intraperitoneal Oral

Intraperitoneal

Results Poor CTL activity, poor memory Poor viral clearance in serum and in peripheral tissues at high doses Same: Either presence of LTαβ on activated T-cells or organized lymphoid microrarchitecture/intact MZ are required for responses Reduced numbers of LCMV-specific CD8⫹ lymphocytes Reduced IFN-γ secretion by LCMV-specific CD8⫹ lymphocytes LTα⫺/⫺ CD8⫹ T-cells responded normally to LCMV

Improved survival and prevention of pulmonary distress Reduced numbers of LCMV-specific CD8⫹ lymophcytes Enhanced mortality and development of lesions and/or encephalitis Normal numbers of HSV-specific CD8⫹ T-cells Reduced IFN-γ secretion by HSV-specific CD8⫹ T-cells Modest intrinsic defect in CTL-activity of LTα⫺/⫺ CD8⫹ T-cells Reduced clearance only at high dose Low-dose clearance delayed but normal Delayed appearance of influenza-specific CD8⫹ T-cells Normal IFN-γ secretion and CTL activity at low doses of influenza Normal clearance Impaired clearance and granuloma formation and enhanced mortality Reduced survival and poor mycrobacterial containment Impaired granuloma formation in the spleen and reduced IFN-γ Poor survival Failure to control infection, poor survival, delayed appearance of T. Gondii specific T-cells and IFN-γ secreting T-cells Normal response, improved survival

Note: Listed here are infections disease models evaluated in LTαβ/LIGHT pathway inhibited rodents. For simplicity, only cases that are relevant to T-cell biology (and not lymph node development) have been listed. In addition, only models that are affected by inhibition the LTαβ/LIGHT pathway but not the TNFα/LTα pathway are discussed.

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results in susceptibility of LTα ⫺/⫺ mice to development of lesions and/or encephalitis and enhanced mortality compared to control mice. In spite of normal numbers of HSV-specific CD8⫹ T-cells in LTα ⫺/⫺ mice, these CD8⫹ T-cells exhibited reduced IFN-γ secretion and diminished CTL responses. Interestingly, the intrinsic CTL activity of LTα ⫺/⫺ CD8⫹ T-cells to antigen as assessed in vitro was modestly reduced. This particular experiment compared naı¨ve LTα ⫺/⫺ versus WT lymphocytes stimulated with peptide-pulsed targets. Therefore, this is one of the first examples that demonstrates that absence of LTα on T-cells leads to an intrinsic T-cell defect that is independent of the microenvironment. In contrast to these studies, Lund et al. [82] examined the effect of influenza type A infection on LTα ⫺/⫺ mice. As mentioned before, LTα ⫺/⫺ mice do not have LN, but interestingly, these mice have intact NALT, albeit greatly reduced in size. Thus, unlike GALT, NALT development is LT-independent. Since the route of influenza A administration was intranasal, it was of interest to determine if NALT could compensate for the absence of LN. In this study, LTα ⫺/⫺ mice were more susceptible to influenza A infection at high doses, but mice infected with low doses of virus were unaffected by the absence of LTα and LN. The appearance of influenza-specific CD8⫹ T-cells was delayed in LTα ⫺/⫺ mice by three days, but the magnitude of the response was comparable. Accordingly, viral clearance was delayed in LTα ⫺/⫺ mice, but virus was eventually cleared. Unlike the other viral studies, IFN-γ secretion by CD8⫹ T-cells and CTL activity were not impaired in LTα ⫺/⫺ mice, at least at relatively low infectious doses of influenza-A [82]. Therefore, this study demonstrated that there was not an obvious defect in influenza responses at low doses of the pathogen in LTα ⫺/⫺ mice. Some of these read-outs were not tested at higher doses of pathogen, and this might explain the discrepancy between this study and the other viral infection models. Furthermore, the route of infection (intranasal vs. intradermal vs. systemic) likely influences whether LTαβ/LIGHT pathway is required for antigen-specific responses, and the nature of the pathogen and relative virulence of the pathogen are also important factors. Indeed, similar discrepancies have been encountered in analysis of pathogen responses in the context of other TNF family members such as the 41BB pathway (reviewed in [83]). 5.3. Intracellular bacterial infections Intracellular bacterial infections have been tested in LTα⫺, LTβR and LTβ-deficient mice. A thorough study was performed by Roach et al. [84] where the LTα⫺ and LTβ-deficient mice were derived from the C57BL/6 background. Bone marrow chimeras were then made with these mice so that the chimeras were LN competent. These chimeras were infected with Mycobacterium tuberculosis by aerosol inoculation, and their survival was monitored. LTβ ⫺/⫺ mice resolved their bacterial infections in a manner comparable to wild type mice, whereas LTα⫺/⫺ mice could not control the infection and exhibit defective granuloma formation [84]. Therefore, LTα homotrimer rather than LTαβ heterotrimer is important for resolving M. tubercuolosis infection. A different study by Lucas et al. [85] examined the effect of LTßR-Ig treatment on M. bovis infection. In this case, infection was systemic as immunization was by the intravenous route. Lucas et al. [85] found that there was a decrease in granuloma formation in the spleen, but not the lungs of infected LTßR-Ig treated mice and there was a concomitant decrease in IFN-γ but up-regulation of IL-4 in the serum of these mice.

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Therefore, whereas Roach et al. [84] show that LTβ ⫺/⫺ mice are not susceptible to intracellular bacterial infection, this study implicates LTαβ and/or LIGHT mediated signaling in the formation of granuloma in the spleen, at least for systemic administration of bacteria [85]. In agreement with a role for LTαβ pathway in controlling M. tuberculosis, Ehlers et al. [86] showed that LTα and LTβ but not LIGHT-deficient mice exhibit impaired bacterial clearance from the liver and lung tissues after intranasal infection, and decreased survival in LTßR ⫺/⫺ mice. The protective effect of the LTαβ pathway was postulated to be via signaling of LTßR on pulmonary macrophages, and irradiated WT mice that received BM from LTßR-deficient donors showed similar susceptibility to M. tuberculosis. However, a contribution of other bone marrow derived LTßR-expressing cell types such as DC was not ruled out. It is presumed that T-cells provide LTαβ ligand either directly to macrophages or to DC that then influence macrophage activation since T-cells were found to be present within granulomatous lesions [86]. These conflicting studies may perhaps be explained by the route of immunization of bacteria, and perhaps LTßR may have a particular function in regulating different mesenchymal cells, macrophages or DC within different tissues. The same authors showed that LTßR ⫺/⫺ mice are susceptible to intraperitoneal immunization with Listeria monocytogenes, thus presumably LTßR signaling is critical for peritoneal macrophage or DC activation, although this was not directly demonstrated [86]. 5.4. Parasitic infections Susceptibility to Toxoplasma gondii was also examined in TNFα/LTα double knock-out versus LTα and TNFα single knock-out mice. In this study, there was decreased survival in the LTα ⫺/⫺ and TNFα/LTα ⫺/⫺ mice compared to WT and TNFα ⫺/⫺ mice. The frequency of T. gondii specific T-cells was initially reduced in LTα ⫺/⫺ and TNFα/LTα ⫺/⫺ mice, although numbers of specific T-cells recovered later in the infection. Only mice lacking LTα exhibited a defect in the number of IFN-γ secreting T-cells in the spleen. Bone marrow chimeras confirmed that the absence of LTα on hematopoietic cells was required for susceptibility to T. gondii [87]. In contrast, LTα ⫺/⫺ mice are capable of controlling Trypanosoma brucei infections effectively, and have improved survival compared to WT mice [88]. These differences may reflect the differential requirement for IFN-γ in controlling parasitic infiltration. In summary, infection with a wide range of pathogens has led to mixed results in the effects of LTαβ/LIGHT pathway inhibition on clearance of infectious organisms. The different routes of infection, dose of virus and configuration of the host genes accounts for much of this variability (Table 2.). Systematic comparisons of similar antigens using different routes of administration and multiple endpoints at different time points will be required to unravel this confusing picture.

6. Dissecting a mechanism of action for why LT inhibition prevents autoimmunity The published body of literature indicates that LTαβ/LIGHT pathway is important in autoimmune processes, clearance of some pathogens, maintenance of lymphoid microenvironments and the formation of ectopic tertiary lymphoid structures. Because of the pleiotropic

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effects of this pathway (Fig. 3.), it has been challenging to understand exactly why an agent like LTßR-Ig is effective in such an impressive array of autoimmune models. The study of how LTßR-signaling pathway affects lymphocyte responses has been confounded by the fact that LTßR signaling is required for the constitutive expression of chemokines that are critical for the organization of immune cells, but also that the inducible expression of addressins and other chemokines that are important at sites of chronic inflammation are influenced by the LTßR-signaling pathway [48,64,89]. Moreover, the up-regulation of LTαβ and LIGHT on activated lymphocytes has barely been addressed independent of the microenvironment. Using antigen-specific systems, one can obtain more refined information about the effect of LTαβ/LIGHT pathway on the activation and maturation of T-cell responses. Suresh et al. [79] examined LCMV-specific CD8⫹ T-cells in LCMV-infected LTα ⫺/⫺ mice using tetramer reagents that detect T-cells specific for a particular MHC/peptide combination. In this study, there was a decrease in LCMV-specific T-cells in these mice, but there was an even greater reduction in IFN-γ secreting CD8⫹ LCMV-specific T-cells [79]. Kumuraguru et al. [81] also demonstrated that the number of HSV-specific T-cells is not affected in LTα ⫺/⫺ mice, but that these cells exhibited decreased IFN-γ production. In the relapsing-remitting EAE model, LTßR-Ig did not prevent acute phase disease, suggesting normal priming and expansion of encephalogenic T-cells, but relapses were strongly impaired. T-cells tested ex vivo for recall responses to encephalogenic peptide proliferated poorly and failed to secrete significant amounts of IFN-γ [74]. Therefore, a common theme is emerging that LTαβ/ LIGHT pathway may have a role in the generation of a mature effector T-cell response rather than influencing initial early T-cell responses. In agreement with this hypothesis, expansion of transgenic T-cells in response to subcutaneous immunization with ovalbumin peptide in adjuvant was found to be normal in LTßR-Ig treated mice, but recall responses to the peptide in vitro were impaired [74]. In these studies, systemic, intradermal or subcutaneous routes were used to administer antigen. In contrast, the study by Lund et al. [82] on influenza A infection used an intranasal route of infection of LTα ⫺/⫺ mice, and although there is a slight delay in responsiveness to influenza A, there is no defect in IFN-γ secretion by influenza A-specific LTα ⫺/⫺ T-cells. This raises the interesting possibility that LTαβ and/or LIGHT pathway may play varying roles at different anatomical sites, and the lung compartment may not be as dependent on LT/LIGHT signals as the peripheral draining LN. However, the influenza A results are in contrast to a recent publication by Lo et al. [90] where they showed that CCL21 expression can be up-regulated in a LT-dependent fashion following a strong airway challenge with soluble egg antigen from Schistosoma mansoni. These authors made the connection that this sustained CCL21 expression in the lung is required for further homing of lymphocytes to this site of inflammation [90]. It is likely that not only the site of antigen-encounter but also the strength of the antigenic challenge will be variables that influence whether LTαβ/ LIGHT pathway is required for effective T-cell responses. For these reasons, it will be interesting to compare and contrast the contribution of LTαβ/LIGHT pathway in systemic immune responses versus immune responses in peripheral or mucosal sites. Initially, a role for LTαβ pathway in controlling humoral responses was ascribed because FDC presence and antigen-trapping function were strongly affected in LT deficient and LTßRIg treated animals [16,17]. However, a role for FDC in sustaining humoral responses has

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been questioned in a recent study [18,19]. Moreover, the dose of antigen likely has an important influence on whether FDC are required for the humoral response. In the collagen-induced arthritis model, LTßR-Ig treatment blunted the development of pathogenic anticollagen titers and it was unclear whether T-cells were affected in this model [77]. Therefore, there is likely a therapeutic benefit for using LTßR-Ig to curtail autoantibody responses. This may be due to its effects at dissolving ectopic lymphoid structures, as GC in these structures have been correlated with disease, or alternatively due to poor T-cell help provided to antibody secreting B-cells. There are two key confounding factors to interpreting all of these data. One is that many of these studies were performed in LTαβ pathway knock-out mice that have profound developmental defects. To get past this complication, two approaches have been taken. The first is to perform bone marrow chimeras where wild type, LN/PP competent mice are reconstituted with LTα or LTβ ⫺/⫺ bone marrow. The second approach is to administer pharmacological reagents that will block LTαβ pathway in the adult animal. However, the affects of LTßR-Ig on the microenvironment are well-characterized [17,25], and chimeric mice also have significant lymphoid microenvironment defects [78]. This brings one to the second problem. Although the absence of LN can be circumvented by using chimeras or pharmacological reagents, all of these studies were executed in the context of a mouse with significantly altered lymphoid microenvironments. The exception to this observation is found in two studies: one in vitro CTL experiment [81], and a short-term adoptive transfer experiment using 100 million donor LTα ⫺/⫺ T-cells [79]. It is feasible that the expression of LTαβ and/or LIGHT on activated CD4⫹ T-cells is required for stimulation of LTßR-expressing DC. This would in turn license the DC to secrete cytokines such as IL-12. This feedback-loop could also have consequences on cross-presentation of antigen in order to generate an effective CD8⫹ T-cell response. Since this hypothesis has not been satisfactorily eliminated in the literature, the employment of transgenic systems where the expression of LTαβ/LIGHT on CD4 cells can be controlled genetically is a necessary and promising approach for unraveling the role of LTαβ/LIGHT on activated lymphocytes from its effect on the local microenvironment.

7. Clinical relevance There are several reasons why inhibition of the LTαβ/LIGHT pathway is a promising therapeutic approach: 1. Efficacy of LTßR-Ig is comparable to that of TNF inhibitors in several rodent autoimmune models such as CIA and IBD. Therefore, for patients who are unresponsive to anti-TNF therapies, LTßR-Ig may represent an advantageous alternative. 2. There are many points in which the biological effects of LTαβ/LIGHT pathway versus TNF/LTα pathway diverge. Notably, constitutive expression of the CCL19, CCL21 and CXCL13 chemokines is significantly more dependent on LTßR signaling rather than TNFR signaling [26]. Moreover, the absence of TNF seems to ameliorate EAE in the early stages, but in the late stages, enhanced T-cell responses are observed [91].

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This is in contrast to the results observed for LTßR-Ig treatment of chronic EAE in SJL mice where relapses rather than acute phase disease are inhibited and ex vivo recall responses at late time points (nine weeks post immunization) are attenuated rather than exacerbated. Therefore, LT and TNF/LTα pathways clearly play different roles in EAE pathogenesis and the examination of EAE models in the absence of PTx has elucidated these differences. Thus, LTαβ/LIGHT inhibition and TNF inhibition may have complementary rather than overlapping mechanisms of action, suggesting that coadministration of both a LTαβ/LIGHT blocker and a TNF blocker may have an additive or synergistic effect in treating autoimmune diseases such as IBD and RA. 3. Membrane-bound LTαβ is expressed at low levels on a subset of resting B-cells [15], but highly expressed on T-lymphocytes activated through the T-cell receptor [4] or via cytokine stimulation [52]. In addition, LTαβ ligand is highly expressed in chronically inflamed tissues in humans [92,93]. Therefore, blocking interactions between LTßR and LTαβ may have a dramatic effect at curtailing inflammatory processes in diseased tissues. 4. LTßR-Ig treatment has been shown to arrest the formation of ectopic lymphoid structures and down-regulate addressins expressed on the HEVs of these structures [49,52]. Since ectopic lymphoid structures and the recruitment of inflammatory cells to these structures has been observed in diseased tissues of autoimmune patients, circumventing this lymphoid neo-organogenesis may have therapeutic benefit. 5. The therapeutic effects of LTßR-Ig were observed at levels as low as 0.1 mg/kg in an acute EAE model [74] and at a serum concentration of two to three µg/ml in the NOD diabetes model [32]. Therefore, it is expected that this inhibitor will be effective at very low doses.

8. Summary Based on the multiple studies using either models of autoimmunity or infectious disease, there is much promise for the use of LTαβ/LIGHT inhibitors in the clinic. However, much remains to be learned about its mechanism of action on inhibiting the activities of autoreactive T-lymphocytes. Moreover, dissecting the contribution of LIGHT-HVEM versus LTαβ-LTßR interactions in autoimmune disease remains poorly elucidated and dissociating the effects of LTßR-Ig on the lymphoid microenvironment versus a more direct effect on LTαβ-expressing activated lymphocytes has not been adequately attempted. Experiments using more refined model systems exploiting transgenic and knock-out mice will undoubtedly yield further information that will shed light on this interesting pathway.

Acknowledgments The author wishes to thank Dr. Jeffrey Browning and Dr. Tania Watts for critical reading of the manuscript. Dr. Gommerman is currently funded by the Canadian Institutes of Health Research for studying the role of the LTαβ/LIGHT pathway in T cell responses.

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References [1] Aggarwal BB, Natarajan K. Tumor necrosis factors: developments during the last decade. Eur Cytokine Netw 1996;7:93–124. [2] Ware CF, Van Arsdale TL, Crowe PD, Browning JL. The ligands and receptors of the lymphotoxin system. Curr Top Microbiol Immunol 1995;198:175–218. [3] Granger SW, Ware CF. Turning on LIGHT. J Clin Invest 2001;108:1741–2. [4] Browning JL, Sizing ID, Lawton P, Bourdon PR, Rennert PD, Majeau GR, et al. Characterization of lymphotoxin-alpha beta complexes on the surface of mouse lymphocytes. J Immunol 1997;159:3288–98. [5] Browning JL, French LE. Visualization of lymphotoxin-beta and lymphotoxin-beta receptor expression in mouse embryos. J Immunol 2002;168:5079–87. [6] Granger SW, Rickett S. LIGHT-HVEM signaling and the regulation of T cell-mediated immunity. Cytokine Growth Factor Rev 2003;14:289–96. [7] Mebius RE. Organogenesis of lymphoid tissues. Nat Rev Immunol 2003;3:292–303. [8] Muller G, Lipp M. Concerted action of the chemokine and lymphotoxin system in secondary lymphoid organ development. Curr Opin Immunol 2003;15:217–24. [9] Scheu S, Alferink J, Potzel T, Barchet W, Kalinke U, Pfeffer K. Targeted disruption of LIGHT causes defects in costimulatory T cell activation and reveals cooperation with lymphotoxin beta in mesenteric lymph node genesis. J Exp Med 2002;195:1613–24. [10] Gommerman JL, Browning JL. Lymphotoxin/LIGHT, lymphoid microenvironments and autoimmune disease. Nat Rev Immunol 2003;3:642–55. [11] Camacho SA, Kosco-Vilbois MH, Berek C. The dynamic structure of the germinal center. Immunol Today 1998;19:511–4. [12] Tew JG, Wu J, Qin D, Helm S, Burton GF, Szakal AK. Follicular dendritic cells and presentation of antigen and costimulatory signals to B cells. Immunol Rev 1997;156:39–52. [13] Martin F, Kearney JF. Marginal-zone B cells. Nat Rev Immunol 2002;2:323–35. [14] Von-Andrian UH, Mempel TR. Homing and cellular traffic in lymph nodes. Nat Rev Immunol 2003;3:867–78. [15] Ansel KM, Ngo VN, Hyman PL, Luther SA, Forster R, Sedgwick JD, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles. Nature 2000;406:309–14. [16] Mackay F, Browning JL. Turning off follicular dendritic cells. Nature 1998;395:26–7. [17] Gommerman JL, Mackay F, Donskoy E, Meier W, Martin P, Browning JL. Manipulation of lymphoid microenvironments in nonhuman primates by an inhibitor of the lymphotoxin pathway. J Clin Invest 2002; 110:1359–69. [18] Martin S, Goodnow C. Immunology. Memory needs no reminders. Nature 2000;407:576–7. [19] Maruyama M, Lam KP, Rajewsky K. Memory B-cell persistence is independent of persisting immunizing antigen. Nature 2000;407:636–42. [20] Koni PA, Sacca R, Lawton P, Browning JL, Ruddle NH, Flavell RA. Distinct roles in lymphoid organogenesis for lymphotoxins alpha and beta revealed in lymphotoxin beta-deficient mice. Immunity 1997;6:491–500. [21] Matsumoto M, Lo SF, Carruthers CJ, Min J, Mariathasan S, Huang G, et al. Affinity maturation without germinal centres in lymphotoxin-alpha-deficient mice. Nature 1996;382:462–6. [22] Futterer A, Mink K, Luz A, Kosco-Vilbois MH, Pfeffer K. The lymphotoxin beta receptor controls organogenesis and affinity maturation in peripheral lymphoid tissues. Immunity 1998;9:59–70. [23] Wang Y, Huang G, Wang J, Molina H, Chaplin DD, Fu YX. Antigen persistence is required for somatic mutation and affinity maturation of immunoglobulin. Eur J Immunol 2000;30:2226–34. [24] Ettinger R, Browning JL, Michie SA, Van Ewijk W, McDevitt HO. Disrupted splenic architecture, but normal lymph node development in mice expressing a soluble lymphotoxin-beta receptor-IgG1 fusion protein. Proc Natl Acad Sci USA 1996;93:13102–7. [25] Mackay F, Majeau GR, Lawton P, Hochman PS, Browning JL. Lymphotoxin but not tumor necrosis factor functions to maintain splenic architecture and humoral responsiveness in adult mice. Eur J Immunol 1997;27:2033–42. [26] Ngo VN, Korner H, Gunn MD, Schmidt KN, Sean Riminton D, Cooper MD, et al. Lymphotoxin alpha/ beta and tumor necrosis factor are required for stromal cell expression of homing chemokines in B and T cell areas of the spleen. J Exp Med 1999;189:403–12.

390

J. Gommerman /Clin. Applied Immunol. Rev. 4 (2004) 367–393

[27] Luther SA, Tang HL, Hyman PL, Farr AG, Cyster JG. Coexpression of the chemokines ELC and SLC by T zone stromal cells and deletion of the ELC gene in the plt/plt mouse. Proc Natl Acad Sci USA 2000;97:12694–9. [28] Gunn MD, Tangemann K, Tam C, Cyster JC, Rosen SD, Williams LT. A chemokine expressed in lymphoid high endothelial venules promotes the adhesion and chemotaxis of naive T lymphocytes. Proc Natl Acad Sci USA 1998;95:258–63. [29] Cyster JG. Leukocyte migration: scent of the T zone. Curr Biol 2000;10:R30–3. [30] Wu Q, Wang Y, Wang J, Hedgeman EO, Browning JL, Fu YX. The requirement of membrane lymphotoxin for the presence of dendritic cells in lymphoid tissues. J Exp Med 1999;190:629–38. [31] Korner H, Winkler TH, Sedgwick JD, Rollinghoff M, Basten A, Cook MC. Recirculating and marginal zone B cell populations can be established and maintained independently of primary and secondary follicles. Immunol Cell Biol 2001;79:54–61. [32] Ettinger R, Munson SH, Chao CC, Vadeboncoeur M, Toma J, McDevitt HO. A critical role for lymphotoxinbeta receptor in the development of diabetes in nonobese diabetic mice. J Exp Med 2001;193:1333–40. [33] Lu TT, Cyster JG. Integrin-mediated long-term B cell retention in the splenic marginal zone. Science 2002;297:409–12. [34] Mandik-Nayak L, Huang G, Sheehan KC, Erikson J, Chaplin DD. Signaling through TNF receptor p55 in TNF-alpha-deficient mice alters the CXCL13/CCL19/CCL21 ratio in the spleen and induces maturation and migration of anergic B cells into the B cell follicle. J Immunol 2001;167:1920–8. [35] Pasparakis M, Alexopoulou L, Episkopou V, Kollias G. Immune and inflammatory responses in TNFα deficient mice: a critical requirement for TNFα in the formation of primary B cell follicles, follicular dendritic cell networks and germinal centers and in the maturation of the humoral immune response. J Exp Med 1996;184:1397–411. [36] Boehm T, Scheu S, Pfeffer K, Bleul CC. Thymic medullary epithelial cell differentiation, thymocyte emigration, and the control of autoimmunity require lympho-epithelial cross talk via LTBR. J Exp Med 2003;5:757–69. [37] Chin RK, Lo JC, Kim O, Blink SE, Christiansen PA, Peterson P, et al. Lymphotoxin pathway directs thymic Aire expression. Nat Immunol 2003;4:1121–7. [38] Kajiura F, Sun S, Nomura T, Izumi K, Ueno T, Bando Y, et al. NF-kappaB-inducing kinase establishes self-tolerance in a thymic stroma-dependent manner. J Immunol 2004;172:2067–75. [39] Debard N, Sierro F, Browning JL, Kraehenbuhl JP. Effect of mature lymphoctes and lymphotoxin on the development of the follicle-associated epithelium and M cells in mouse Peyer’s patches. Gastroenterology 2001;167:2781–90. [40] Dohi T, Rennert PD, Fujihashi K, Kiyono H, Shirai Y, Kawamura YI, et al. Elimination of colonic patches with lymphotoxin beta receptor-Ig prevents Th2 cell-type colitis. J Immunol 2001;167:2781–90. [41] Lorenz RG, Chaplin DD, McDonald KG, McDonough JS, Newberry RD. Isolated lymphoid follicle formation is inducible and dependent upon lymphotoxin-sufficient B lymphocytes, lymphotoxin beta receptor, and TNF receptor I function. J Immunol 2003;170:5475–82. [42] Chin R, Wang J, Fu Y-X. Lymphoid microenvironment in the gut for immunoglobulin A and inflammation. Immunol Rev 2003;195:190–201. [43] Kang HS, Chin RK, Wang Y, Yu P, Wang J, Newell KA, et al. Signaling via LTbetaR on the lamina propria stromal cells of the gut is required for IgA production. Nat Immunol 2002;3:576–82. [44] Zinkernagel RM. Localization dose and time of antigens determine immune reactivity. Semin Immunol 2000;12:163–71. [45] Weyand CM, Kurtin PJ, Goronzy JJ. Ectopic lymphoid organogenesis, a fast track for autoimmunity. Am J Pathol 2001;159:787–93. [46] Van Nierop K, de Groot C. Human follicular dendirtic cells: function, origin and development. Semin Immunol 2002;14:251–7. [47] Kratz A, Campos-Neto A, Hanson MS, Ruddle NH. Chronic inflammation caused by lymphotoxin is lymphoid neogenesis. J Exp Med 1996;183:1461–72. [48] Drayton DL, Ying X, Lee J, Lesslauer W, Ruddle NH. Ectopic LTab directs lymphoid organ neogenesis with concomitant expression of peripheral node addressin and a HEV-restricted sulfotransferase. J Exp Med 2003;197:1153–63.

J. Gommerman /Clin. Applied Immunol. Rev. 4 (2004) 367–393

391

[49] Luther SA, Lopez T, Bai W, Hanahan D, Cyster JG. BLC expression in pancreatic islets causes B cell recruitment and lymphotoxin-dependent lymphoid neogenesis. Immunity 2000;12:471–81. [50] Chen SC, Vassileva G, Kinsley D, Holmann S, Manfra D, Wiekowski MT, et al. Ectopic expression of the murine chemokines CCL21a and CCL21b induces the formation of lymph node-like structures in the pancreas, but not the skin, of transgenic mice. J Immunol 2002;168:1001–8. [51] Fan L, Reilly CR, Luo Y, Dorf ME, Lo D. Cutting edge: ectopic expression of the chemokine TCA4/SLC is sufficient to trigger lymphoid neogenesis. J Immunol 2000;164:3955–9. [52] Luther SA, Bidgol A, Hargreaves DC, Schmidt A, Xu Y, Paniyadi J, et al. Differing activities of homeostatic chemokines CCL19, CCL21, and CXCL12 in lymphocyte and dendritic cell recruitment and lymphoid neogenesis. J Immunol 2002;169:424–33. [53] Spahn TW, Kucharzik T. Modulating the intestinal immune system: the role of lymphotoxin and GALT organs. Gut 2004;53:456–65. [54] Shaikh RB, Santee S, Granger SW, Butrovich K, Cheung T, Kronenberg M, et al. Constitutive expression of LIGHT on T cells leads to lymphocyte activation, inflammation, and tissue destruction. J Immunol 2001; 167:6330–7. [55] Wang J, Lo JC, Foster A, Yu P, Chen HM, Wang Y, et al. The regulation of T cell homeostasis and autoimmunity by T cell-derived LIGHT. J Clin Invest 2001;108:1771–80. [56] Stopfer P, Obermeier F, Dunger N, Falk W, Farkas S, Janotta M, et al. Blocking lymphotoxin-beta receptor activation diminishes inflammation via reduced mucosal addressin cell adhesion molecule-1 (MAdCAM1) expression and leucocyte margination in chronic DSS-induced colitis. Clin Exp Immunol 2004;136:21–9. [57] Mackay F, Browning JL, Lawton P, Shah SA, Comiskey M, Bhan AK, et al. Both the lymphotoxin and tumor necrosis factor pathways are involved in experimental murine models of colitis. Gastroenterology 1998;115:1464–75. [58] Wu Q, Salomon B, Chen M, Wang Y, Hoffman LM, Bluestone JA, et al. Reversal of spontaneous autoimmune insulitis in nonobese diabetic mice by soluble lymphotoxin receptor. J Exp Med 2001;193:1327–32. [59] Sung H-H, Juang J-H, Lin Y-C, Kuo C-H, Hung J-T, Chen A, et al. Transgenic expression of decoy receptor 3 protects islets from spontaneous and chemical-induced autoimmune destruction in nonobese diabetic mice. J Exp Med 2004;199:1143–51. [60] Tamada K, Shimozaki K, Chapoval AI, Zhai Y, Su J, Chen SF, et al. LIGHT, a TNF-like molecule, costimulates T cell proliferation and is required for dendritic cell-mediated allogeneic T cell response. J Immunol 2000;164:4105–10. [61] Tamada K, Shimozaki K, Chapoval AI, Zhu G, Sica G, Flies D, et al. Modulation of T-cell-mediated immunity in tumor and graft-versus-host disease models through the LIGHT co-stimulatory pathway. Nat Med 2000;6:283–9. [62] Tamada K, Tamura H, Flies D, Fu YX, Celis E, Pease LR, et al. Blockade of LIGHT/LTbeta and CD40 signaling induces allospecific T cell anergy, preventing graft-versus-host disease. J Clin Invest 2002; 109:549–57. [63] Wu Q, Fu YX, Sontheimer RD. Blockade of lymphotoxin signaling inhibits the clinical expression of murine graft-versus-host skin disease. J Immunol 2004;172:1630–6. [64] Guo Z, Wang J, Meng L, Wu Q, Kim O, Hart J, et al. Cutting edge: Membrane lymphotoxin regulates CD8(⫹) T cell-mediated intestinal allograft rejection. J Immunol 2001;167:4796–800. [65] Zhang J, Salcedo TW, Wan X, Ullrich S, Hu B, Gregorio T, et al. Modulation of T-cell responses to alloantigens by TR6/DcR3. J Clin Invest 2001;107:1459–68. [66] Ye Q, Fraser CC, Gao W, Wang L, Busfield SJ, Wang C, et al. Modulation of LIGHT-HVEM costimulation prolongs cardiac allograft survival. J Exp Med 2002;195:795–800. [67] Frei K, Eugster H-P, Bopst C, Constantinescu C, Lavi E, Fontana A. Tumor necrosis factor α and lymphotoxin α are not requied for induction of acute experimental autoimmune encephalomyelitis. J Exp Med 1997;185:2177–82. [68] Korner H, Riminton DS, Strickland DH, Lemckert FA, Pollard JD, Sedgwick JD. Critical points of tumor necrosis factor action in central nervous system autoimmune inflammation defined by gene targeting. J Exp Med 1997;186:1585–90.

392

J. Gommerman /Clin. Applied Immunol. Rev. 4 (2004) 367–393

[69] Riminton DS, Korner H, Strickland DH, Lemckert FA, Pollard JD, Sedgwick JD. Challenging cytokine redundancy: Inflammatory cell movement and clinical course of experimental autoimmune encephalomyelitis are normal in lymphotoxin-deficient, but not tumor necrosis factor-deficient mice. J Exp Med 1998;187: 1517–28. [70] Suen WE, Bergman CM, Hjelmstrom P, Ruddle NH. A critical role for lymphotoxin in experimental allergic encephalomyelitis. J Exp Med 1997;186:1233–40. [71] Browning JL, Ngam-ek A, Lawton P, DeMarinis J, Tizard R, Chow EP, et al. Lymphotoxin beta, a novel member of the TNF family that forms a heteromeric complex with lymphotoxin on the cell surface. Cell 1993;72:847–56. [72] Steinman L. Some misconceptions about understanding autoimmunity through experiments with knockouts. J Exp Med 1997;185:2039–41. [73] Cyster JG, Goodnow CC. Pertussis toxin inhibits migration of B and T lymphocytes into splenic white pulp cords. J Exp Med 1995;182:581–6. [74] Gommerman JL, Giza K, Perper S, Sizing I, Ngam-ek A, Nickerson-Nutter C, et al. A role for surface lymphotoxin in experimental autoimmune encephalomyelitis independent of LIGHT. J Clin Invest 2003; 112:755. [75] Goluszko E, Hjelmstrom P, Deng C, Poussin MA, Ruddle NH, Christadoss P. Lymphotoxin-alpha deficiency completely protects C57BL/6 mice from developing clinical experimental autoimmune myasthenia gravis. J Neuroimmunol 2001;113:109–18. [76] Shao H, Fu Y, Song L, Sun S, Kaplan HJ, Sun D. Lymphotoxin beta receptor-Ig fusion protein treatment blocks actively induced, but not adoptively transferred, uveitis in Lewis rats. Eur J Immunol 2003;33:1736–43. [77] Fava RA, Notidis E, Hunt J, Szanya V, Ratcliffe N, Ngam-ek A, et al. A role for the lymphotoxin/LIGHT axis in the pathogenesis of murine collagen induced arthritis. J Immunol 2003;171:115–26. [78] Berger DP, Naniche D, Crowley MT, Koni PA, Flavell RA, Oldstone MB. Lymphotoxin-beta-deficient mice show defective antiviral immunity. Virology 1999;260:136–47. [79] Suresh M, Lanier G, Large MK, Whitmire JK, Altman JD, Ruddle NH, et al. Role of lymphotoxin alpha in T-cell responses during an acute viral infection. J Virol 2002;76:3943–51. [80] Puglielli MT, Browning JL, Brewer AW, Schreiber RD, Shieh WJ, Altman JD, et al. Reversal of virus-induced systemic shock and respiratory failure by blockade of the lymphotoxin pathway. Nat Med 1999;5:1370–4. [81] Kumaraguru U, Davis IA, Deshpande S, Tevethia SS, Rouse BT. Lymphotoxin alpha⫺/⫺ mice develop functionally impaired CD8⫹ T cell responses and fail to contain virus infection of the central nervous system. J Immunol 2001;166:1066–74. [82] Lund FE, Partida-Sanchez S, Lee BO, Kusser KL, Hartson L, Hogan RJ, et al. Lymphotoxin-alpha-deficient mice make delayed, but effective, T and B cell responses to influenza. J Immunol 2002;169:5236–43. [83] Bertram EM, Dawicki W, Watts TH. Role of T cell costimulation in anti-viral immunity. Semin Immunol 2004;16:185–96. [84] Roach DR, Briscoe H, Saunders B, France MP, Riminton S, Britton WJ. Secreted lymphotoxin-alpha is essential for the control of an intracellular bacterial infection. J Exp Med 2001;193:239–46. [85] Lucas R, Tacchini-Cottier F, Guler R, Vesin D, Jemelin S, Olleros ML, et al. A role for lymphotoxin beta receptor in host defense against Mycobacterium bovis BCG infection. Eur J Immunol 1999;29:4002–10. [86] Ehlers S, Holscher C, Scheu S, Tertilt C, Hehlgans T, Suwinski J, et al. The lymphotoxin beta receptor is critically involved in controlling infections with the intracellular pathogens Mycobacterium tuberculosis and Listeria monocytogenes. J Immunol 2003;170:5210–8. [87] Schluter D, Kwok LY, Lutjen S, Soltek S, Hoffmann S, Korner H, et al. Both lymphotoxin-alpha and TNF are crucial for control of Toxoplasma gondii in the central nervous system. J Immunol 2003;170:6172–82. [88] Magez S, Stijlemans B, Caljon G, Eugster HP, De Baetselier P. Control of experimental Trypanosoma brucei infections occurs independently of lymphotoxin-alpha induction. Infect Immun 2002;70:1342–51. [89] Yu P, Lee Y, Liu W, Chin RK, Wang J, Wang Y, et al. Priming of naive T cells inside tumors leads to eradication of established tumors. Nat Immunol 2004;5:141–9. [90] Lo JC, Chin RK, Lee Y, Hyung-Sik K, Wang Y, Weinstock JV, et al. Differential regulation of CCL21 in lymphoid/nonlymphoid tissues for effectively attracting T cells to peripheral tissues. J Clin Invest 2003;112:1495–505.

J. Gommerman /Clin. Applied Immunol. Rev. 4 (2004) 367–393

393

[91] Kassiotis G, Kollias G. Uncoupling the proinflammatory from the immunosuppressive properties of tumor necrosis factor (TNF) at the p55 TNF receptor level: Implications for pathogenesis and therapy of autoimmune demyelination. J Exp Med 2001;193:427–34. [92] Buckle GJ, Hollsberg P, Hafler DA. Activated CD8⫹ T cells in secondary progressive MS secrete lymphotoxin. Neurology 2003;60:702–5. [93] Agyekum S, Church A, Sohail M, Krausz T, Van-Noorden S, Polak J, et al. Expression of lymphotoxinbeta (LT-beta) in chronic inflammatory conditions. J Pathol 2003;199:115–21.